A Broken α-Helix in Folded α-Synuclein

Abstract

␣-Synuclein is a small cytosolic protein of presynaptic nerve terminals composed of seven 11-residue repeats and a hydrophilic tail. ␣-Synuclein misfolding and dysfunction may contribute to the pathogenesis of Parkinson's disease and neurodegenerative dementias, but its normal folding and function are unknown. In solution, ␣-synuclein is natively unstructured but assumes an ␣-helical conformation upon binding to phospholipid membranes. We now show that this conformation of ␣-synuclein consists of two ␣-helical regions that are interrupted by a short break. The structural organization of the ␣-helices of ␣-synuclein was not anticipated by sequence analyses and may be important for its pathogenic role.In recent years, the presynaptic protein ␣-synuclein has attracted much attention because of its involvement in neurodegenerative diseases (1-3). Two independent mutations in human ␣-synuclein cause familial Parkinson's disease, and wild type ␣-synuclein is a major component of Lewy bodies, cytoplasmic inclusion bodies found in Parkinson's disease and in several forms of neurodegenerative dementia. However, independent of its role in neurodegenerative diseases, ␣-synuclein is an interesting protein in its own right. It is an abundant presynaptic protein that may regulate neurotransmitter release and may contribute to synaptic plasticity (4 -6). ␣-Synuclein is the founding member of a protein family that additionally includes ␤-and ␥-synucleins and synoretin (7-9). The sequences of all synucleins are similar, although only ␣-synuclein is implicated in disease. Synucleins are composed of six copies (␤-synuclein) or seven copies (all other synucleins) of an unusual 11-residue imperfect repeat, followed by a variable short hydrophilic tail. Synucleins are soluble, natively unfolded proteins that avidly bind to negatively charged phospholipid membranes and become ␣-helical upon binding (10). Although secondary structure predictions indicate that the synuclein repeats could form an amphipathic structure consistent with lipid binding, the ␣-helical conformation is puzzling because the synuclein repeats are punctuated by central glycine residues. Furthermore, in Lewy bodies ␣-synuclein is thought to be in a ␤-strand aggregate, but aggregation of ␣-synuclein into dimers and multimers is promoted by lipid environments that induce an ␣-helical conformation (11-13). In the present study, we have examined the conformation of ␣-synuclein in lipidic environments to understand the relation of its sequence to its physicochemical properties and to map a potential pathway of misfolding in neurodegenerative disease. EXPERIMENTAL PROCEDURESProduction of ␣-Synuclein-Recombinant ␣-synuclein was expressed in bacteria as GST-fusion proteins with a TEV protease recognition sequence preceding the N-terminal methionine and cleaved with TEV protease (Invitrogen), resulting in a single additional glycine residue at the N terminus. After TEV cleavage, ␣-synuclein was isolated as the only heat-stable component upon boiling for 15 min, purified by i...

Structure of membrane-bound α-synuclein studied by site-directed spin labeling

“…These repeats are similar to those found in apolipoproteins, and it was proposed that the lipid interaction of ␣-synuclein could be similar to that of the apolipoproteins (8,13). The involvement of the N terminus in membrane interaction was subsequently confirmed experimentally by analysis of ␣-synuclein deletion mutants (15) and NMR studies of liposomebound ␣-synuclein (18)(19)(20). The latter studies revealed an ordering of the N-terminal repeat regions induced upon membrane binding whereas the highly charged C terminus remained unstructured and, therefore, was not involved in membrane interaction.…”

19F NMR studies of α‐synuclein‐membrane interactions

“…2(B)] are consistent both with this conclusion and with conclusions based on 15 N NMR data, which show that the N-terminal region of the protein binds SDS while the C-terminal region remains disordered. 13,20,24,28,31,32 Solution NMR is a powerful tool for accessing processes that occur over a range of timescales. 26 In 250 mM NaCl, SDS forms larger rod-like micelles, decreasing the tumbling rate of the a-synuclein-micelle complex.…”

“…[21][22][23][24][25] The last $40 residues lack defined structure and do not appear to be involved in membrane interactions. 13,21 Recent solution NMR data, however, appear incompatible with this model. More specifically, 15 N intensity and relaxation data from titration of SUVs sugget there exists several binding modes in which the first 25 residues adopt a helical state that anchor the interaction with SUVs.…”

“…[8][9][10][11][12] The protein binds lipids and anionic detergents through the seven imperfect, cationic, 11-amino acid repeats located in its N-terminal and hydrophobic regions. [6][7][8][13][14][15][16][17][18][19][20] Electron paramagnetic resonance (EPR) data on its complex with small unilamellar vesicles (SUVs) suggest that the first $100 residues of the monomeric protein adopt an a-helical conformation that lies on the membrane surface. [21][22][23][24][25] The last $40 residues lack defined structure and do not appear to be involved in membrane interactions.…”

“…More specifically, 15 N intensity and relaxation data from titration of SUVs sugget there exists several binding modes in which the first 25 residues adopt a helical state that anchor the interaction with SUVs. 26,27 A variety of other techniques, including circular dichroism spectropolarimetry, 12,13,19,28 fluorescence spectroscopy, 21,[29][30][31] and EPR [21][22][23][24][25] have been used to study how the phospholipid composition of vesicles affects a-synuclein affinity. Although these studies indicate that the properties of phospholipid membranes (i.e., charge, curvature, size, and the identity of the acyl chains) affect binding, there is no consensus, and some studies are contradictory.…”